European Journal of Plastic Surgery

, Volume 37, Issue 6, pp 319–326 | Cite as

Sequencing of the interferon regulatory factor 6 (IRF6) gene and correlation to its phenotypes in familial non-syndromic cleft lip and palate in North Indian population

  • Rohit Khanna
  • Tripti Tikku
  • Gulshan Subodh Jain
  • Kamna Srivastava
  • Mahendra P Darokar
  • Raj Kumar Mishra
Original Paper



The incidence of orofacial clefting varies depending upon geographical location, ethnicity, sex and even socioeconomic status. The study aimed to test the contribution of interferon regulatory factor 6 (IRF6) gene allelic variation to the incidence of non-syndromic cleft lip and/or cleft palate (CL/P) from the population of North India.


Patients from 12 North Indian families with non-syndromic cleft lip with or without cleft palate (NSCL/P) with at least two members affected were clinically evaluated and tested. DNA was extracted from the blood, and three exonic part of the IRF6 gene (exons 2, 5 and 7) were amplified by PCR. Direct sequencing was performed by PCR product, and sequencing results were compared with the reference DNA sequence of the IRF6 gene.


Three SNPs (rs861019, rs2013162 and rs2235372) in 4 out of 12 families were observed. SNP rs861019 was detected in family no. 4 (F4) around exon 2, SNP 2013162 was detected in family no. 10 (F10) and SNP 2235372 was detected in family nos. 5 and 9 (F5 and F9).


This study demonstrates that the three SNPs within the IRF6 gene are associated with NSCL/P, suggesting IRF6 gene sequence variability and variation in this gene, providing first genetic clue to the NSCL/P phenotype in North Indian families. This is our initial step to demonstrate the involvement of the IRF6 gene in NSCL/P patients in North India. Further extensive study and statistical analysis are needed to validate our result.

Level of Evidence: Level IV, diagnostic study.


Non-syndromic cleft lip with or without cleft palate IRF6 gene SNP (rs861019 rs2013162 rs2235372) Cleft lip and palate 



We are grateful to the family members for their participation in this study, to the staff of the Department of Orthodontics and Dentofacial Orthopedics, BBDCODS, Lucknow and Shushrut Institute of Plastic Surgery Hospital, Lucknow. We also would like to thank the Department of Molecular Bioprospection, Central Institute of Medicinal and Aromatic Plants (CSIR) for taking keen interest in this research topic.

Conflict of interest


Ethical standards

This study has been approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All persons gave their informed consent prior to their inclusion in this study.


  1. 1.
    Marazita ML, Mooney MP (2004) Current concepts in the embryology and genetics of cleft lip and cleft palate. Clin Plast Surg 31(2):125–140PubMedCrossRefGoogle Scholar
  2. 2.
    Croen LA, Shaw GM, Jensvold NG, Harris JA (1991) Birth defects monitoring in California: a resource for epidemiological research. Paediatr Perinat Epidemiol 5:423–27PubMedCrossRefGoogle Scholar
  3. 3.
    Ankola AV, Nagesh L, Hegde P, Karibasappa (2005) Primary dentition status and treatment needs of children with cleft lip and/or palate. J Indian Soc Pedod Prev Dent 23:80–82PubMedCrossRefGoogle Scholar
  4. 4.
    Murthy J, Bhaskar L (2009) Current concepts in genetics of nonsyndromic clefts. Indian J Plast Surg 42:68–81PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Schutte BC, Murray JC (1999) The many faces and factors of orofacial clefts. Hum Mol Genet 8:1853–1859PubMedCrossRefGoogle Scholar
  6. 6.
    Mitchell LE, Risch N (1992) Mode of inheritance of nonsyndromic cleft lip with or without cleft palate. Am J Hum Genet 51(2):323–332PubMedCentralPubMedGoogle Scholar
  7. 7.
    Wyszynski DF, Beaty TH (1996) Review of the role of potential teratogens in the origin of human nonsyndromic oral clefts. Teratology 53(5):309–317PubMedCrossRefGoogle Scholar
  8. 8.
    Cobourne MT (2004) The complex genetics of cleft lip and palate. Eur J Orthod 26:7–16PubMedCrossRefGoogle Scholar
  9. 9.
    Jugessur A, Murray JC (2005) Orofacial clefting: recent insights into a complex trait. Curr Opin Genet Dev 15:270–278PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Stanier P, Moore GE (2004) Genetics of cleft lip and palate: syndromic genes contribute to the incidence of non-syndromic clefts. Hum Mol Genet 1:13Google Scholar
  11. 11.
    Schutte BC, Sander A, Malik M, Murray JC (1996) Refinement of the Van der Woude gene location and construction of a 3.5-Mb YAC contig and STS map spanning the critical region in 1q32-q41. Genomics 36(3):507–514PubMedCrossRefGoogle Scholar
  12. 12.
    Janku P, Robinow M, Kelly T, Bralley R, Baynes A, Edgerton MT et al (1980) The van der Woude syndrome in a large kindred: variability, penetrance, genetic risks. Am J Med Genet 5:117–123PubMedCrossRefGoogle Scholar
  13. 13.
    Shprintzen RJ, Goldberg RB, Sidoti EJ (1980) The penetrance and variable expression of the Vander Woude syndrome: implications for genetic counseling. Cleft Palate J 17(1):52–57PubMedGoogle Scholar
  14. 14.
    Ranta R, Rintala AE (1983) Correlations between microforms of the Van der Woude syndrome and cleft palate. Cleft Palate J 20(2):158–162PubMedGoogle Scholar
  15. 15.
    Kondo S, Schutte BC, Richardson RJ, Bjork BC, Knight AS, Watanabe Y et al (2002) Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes. Nat Genet 32(2):219–220CrossRefGoogle Scholar
  16. 16.
    Wang X, Liu J, Zhang H, Xiao M, Li J, Yang C et al (2003) Novel mutations in the IRF6 gene for Van der Woude syndrome. Hum Genet 113:382–386PubMedCrossRefGoogle Scholar
  17. 17.
    Kayano S, Kure S, Suzuki Y, Kanno K, Aoki Y, Kondo S et al (2003) Novel IRF6 mutations in Japanese patients with Van der Woude Syndrome: two missense mutations (R45Q and P396S) and a 17-kb deletion. Hum Genet 48:622–628CrossRefGoogle Scholar
  18. 18.
    NCBI (2011) Single nucleotide polymorphism, NCBI database (dbSNP), build 126. Accessed 12 Jan 2011
  19. 19.
    Ensembl (2011) Ensembl genome browser. Accessed 12 Jan 2011
  20. 20.
    EMBL-EBI (2011) Multiple sequence alignment programs for DNA or proteins. Accessed 12 Jan 2011
  21. 21.
    Zucchero TM, Cooper ME, Maher BS, Daack-Hirsch S, Nepomu-ceno B, Ribeiro L et al (2004) Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate. N Engl J Med 351:769–780PubMedCrossRefGoogle Scholar
  22. 22.
    Blanton SH, Cortez A, Stal S, Mulliken JB, Finnell RH, Hecht JT (2005) Variation in IRF6 contributes to nonsyndromic cleft lip and palate. Am J Med Genet 137A:259–262PubMedCrossRefGoogle Scholar
  23. 23.
    Ghassibé M, Bayet B, Revencu N, Verellen-Dumoulin C, Gillerot Y, Vanwijck R et al (2005) Interferon regulatory factor-6: a gene predisposing to isolated cleft lip with or without cleft palate in the Belgian population. Eur J Hum Genet 13:1239–1242PubMedCrossRefGoogle Scholar
  24. 24.
    Park JW, Mcintosh I, Hetmanski JB, Jabs EW, Vander Kolk CA, Craig A et al (2007) Association between IRF6 and nonsyndromic cleft lip with or without cleft palate in four populations. Genet Med 9:219–227PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Scapoli L, Palmieri A, Martinelli M, Pezzetti F, Carinci P, Tognon M et al (2005) Strong evidence of linkage disequilibrium between polymorphisms at the IRF6 locus and nonsyndromic cleft lip with or without cleft palate, in an Italian population. Am J Hum Genet 76:180–183PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Srichomthong C, Siriwan P, Shotelersuk V (2005) Significant association between IRF6 820G > A and non-syndromic cleft lip with or without cleft palate in the Thai population. J Med Genet 42:e46PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Morkūnienė A, Steponavičiūtė D, Kasnauskienė J, Kučinskas V (2006) Nucleotide sequence changes in the MSX1 and IRF6 genes in Lithuanian patients with nonsyndromic orofacial clefting. Acta Med Lituanica 13(4):219–225Google Scholar
  28. 28.
    Pegelow M, Peyrard-Janvid M, Zucchelli M, Fransson I, Larson O, Kere J, Larsson C et al (2008) Familial non-syndromic cleft lip and palate—analysis of the IRF6 gene and clinical phenotypes. Eur J Orthod 30:169–175PubMedCrossRefGoogle Scholar
  29. 29.
    Diercks GR, Karnezis TT, Kent DT, Flores C, Su GH, Lee JH, Haddad J Jr (2009) The association between interferon regulatory factor 6 (IRF6) and nonsyndromic cleft lip with or without cleft palate in a Honduran population. Laryngoscope 119(9):1759–1764PubMedCrossRefGoogle Scholar
  30. 30.
    Larrabee YC, Birkeland AC, Kent DT, Flores C, Su GH, Lee JH, Haddad J Jr (2011) Association of common variants, not rare mutations, in IRF6 with nonsyndromic clefts in a Honduran population. Laryngoscope 121(8):1756–1759PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Rohit Khanna
    • 1
  • Tripti Tikku
    • 1
  • Gulshan Subodh Jain
    • 1
  • Kamna Srivastava
    • 1
  • Mahendra P Darokar
    • 2
  • Raj Kumar Mishra
    • 3
  1. 1.Department of Orthodontic and Dentofacial OrthopedicBBDCODSLucknowIndia
  2. 2.Department of Molecular BiologyCIMAPLucknowIndia
  3. 3.Department of Plastic SurgeryShushrut InstituteLucknowIndia

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